We previously reported that heat shock protein 105 (HSP105) is overexpressed in a variety of human cancers, including colorectal, pancreatic and esophageal cancer and has proven to be a novel biomarker for the immunohistochemical detection of these cancers. In the present study, we used HLA-transgenic mice (Tgm) and the peripheral blood mononuclear cells (PBMCs) of colorectal cancer patients to identify HLA-A2 and HLA-A24-restricted HSP105 epitopes, as a means of expanding the application of HSP105-based immunotherapy to HLA-A2- or HLA-A24-positive cancer patients. In addition, we investigated by ex vivo IFN-γ ELISPOT assay whether the HSP105-derived peptide of cytotoxic T cells (CTLs) exists in PBMCs of pre-surgical colorectal cancer patients. We found that four peptides, HSP105 A2-7 (RLMNDMTAV), HSP105 A2-12 (KLMSSNSTDL), HSP105 A24-1 (NYGIYKQDL) and HSP105 A24-7 (EYVYEFRDKL), are potential HLA-A2 or HLA-A24-restricted CTL HSP105-derived epitopes. HSP105-specific IFN-γ-secreting T cells were detected in 14 of 21 pre-surgical patients with colorectal cancer in response to stimulation with these four peptides. Our study raises the possibility that these HSP105 peptides are applicable to cancer immunotherapy in patients with HSP105-expressing cancer, particularly colorectal cancer.